Alkylating agents are among the most effective therapeutic agents currently available to treat different malignancies, and are widely used in the clinic (Katzung, In Basic& Clinical Pharmacology, 7th edition, 1998, Appleton & Lange, Stamford, 881). The high degree of cytotoxicity is attributed to the ability to induce DNA interstrand cross-linking thereby inhibiting replication (Rajski and Williams, Chem Reviews 1998, 98: 2723). Among the alkylating agents, the CNU (chloroethylnitrosourea) series have been widely used clinically to treat brain tumors, colon cancer and lymphomas (DeVita, et al. Cancer Res. 1965, 25; 1876; and Nissen, et al. Cancer 1979, 43: 31), however, their clinical usefulness is limited due to delayed and cumulative bone marrow depression and hepatic toxicity (Panasci, et al. Cancer Res. 1977, 37: 2615; and Gibson and Hickman, Biochem Pharmacol. 1982, 31: 2795).
A series of 1,2-bis(sulfonyl)hydrazine prodrugs (SHPs) with the ability to generate chloroethylating and carbamoylating species, but lacking hydroxyethylating and vinylating species, generated by the CNUs had been developed recently (Sartorelli, et al. see U.S. Pat. No. 6,040,338; U.S. Pat. No. 5,637,619; U.S. Pat. No. 5,256,820; U.S. Pat. No. 5,214,068; U.S. Pat. No. 5,101,072; U.S. Pat. No. 4,849,563; and U.S. Pat. No. 4,684,747. The antitumor activity has been suggested to result from chloroethylating and subsequent cross-linking of DNA (Kohn, In Recent Results in Cancer Research, Eds. Carter, et al., 1981, Springer, Berlin, vol. 76: 141; and Shealy, et al., J Med Chem. 1984, 27: 664). The carbamoylating species (i.e., the isocyanate) can react with thiol and amine functionalities on proteins and inhibit DNA polymerase (Baril, et al. Cancer Res. 1975, 35: 1), the repair of DNA strand breaks (Kann, et al. Cancer Res. 1974, 34: 398) and RNA synthesis and processing (Kann, et al. Cancer Res. 1974, 34: 1982). However, hydroxyethylation of DNA is a carcinogenic and/or mutagenic event (Swenson, et al. J Natl Cancer Inst. 1979, 63: 1469).
1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylaminocarbonyl) hydrazine (VNP40101M), the current lead compound in the SHP series, has lower toxicity to hosts and better anti-tumor activities against the L1210 murine leukemia, L1210/BCNU, L1210/CTX, L1210/MEL (1,3-bis(2-chloroethyl)-1-nitrosourea, cyclophosphamide and melphalan resistant sublines), P388 leukemia, M109 lung carcinoma, B16 melanoma, C26 colon carcinoma and U251 glioma than chloroethylnitrosourea (CNU) derivatives and other SHP analogs (Shyam, et al. J Med Chem. 1999, 42: 941). In addition, VNP40101 M is effective in crossing the blood brain barrier (BBB) and eradicating leukemia cells implanted intracranially (>6.54 log cell kill), rivaling the efficacy of BCNU (Finch, et al. Cancer Biochem Biophys. 2001, 61: 3033). 
The anti-tumor activity of VNP40101M is probably due to the release of 90CE and methyl isocyanate. 90CE further fragments to yield methyl 2-chloroethyldiazosulfone (1) FIG. 1, a relatively specific O6-guanine chloroethylator, producing minimal alkylation of the N7-position of guanine (Penketh, et al. J Med Chem. 1994, 37: 2912; and Penketh, et al. Biochem Pharmacol. 2000, 59: 283). Methyl isocyanate released from VNP40101M has the ability to inhibit various DNA repair enzymes including O6-alkylguanine-DNA alkyltransferase leading to stabilization of the O6-alkylguanine monoalkyl species in DNA, which leads to a larger percentage of interstrand cross-links (Baril, et al. Cancer Res. 1975, 35: 1).
VNP40101M is currently in clinical trials in patients with solid tumors and hematologic malignancies. VNP40101M is not very soluble in aqueous solution; polyethylene glycol (PEG) and ethanol are included in the vehicle of the finished product to promote solubility. Both PEG and ethanol are acceptable vehicles for human use but may cause side effects such as hemolysis and phlebitis at high concentrations, as indicated in animal studies. VNP40101M is very well tolerated in humans and could be given at higher doses, and could in theory produce a higher degree of efficacy, if PEG and ethanol could be eliminated from the vehicle. Therefore, our aim was to synthesize a series of SHPs that (a) were capable of improving its water-solubility and stability in aqueous solution at pH 3 to 9; (b) were capable of forming chloroethylating species; (c) were devoid of hydroxyethylating activity; (d) were capable of forming methyl isocyanate; and (e) were capable of improving pharmacokinetic profiles (e.g., longer half-life in vivo).
The present inventors conceived that water-soluble enzymatically-activated SHPs (I) might satisfy the above conditions. An example of such an SHP would be the phosphate containing derivatives shown in FIG. 2 for the following reasons:                (a) In general, a phosphate-bearing analog, including its salt form may have good water-solubility and stability at neutral pH;        (b) The bioconversion of compounds of general structure I is believed to proceed via alkaline phosphatase (AP) cleavage of the oxygen-phosphorous bond to form the phenol intermediate, which may subsequently undergo fragmentation resulting in the formation of chloroethylating or methylating species and carbamoylating agent without generating hydroxyethylating agent, as shown in FIGS. 1 and 2.        
(c) The bioconversion of compounds I may also generate a quinone methide which itself can cause damage to DNA and thereby contribute to inhibition of cellular replication (Lin, et al. J Med Chem. 1986, 29: 84).
(d) Compounds I may be considered as prodrugs of VNP40101M that has been identified as an alkylating agent against a broad anticancer spectrum of neoplastic disease states, including, for example, numerous solid tumors. Thus, compounds I may generate the same active species as VNP40101M.
Further examples of bioactivated prodrugs are shown in FIGS. 3 and 4. The nitro analogs shown in FIG. 3 are examples of compounds that would be both water soluble and selectively activated under conditions of hypoxia. Release of VNP400101M would only occur on reduction of the nitro group under conditions of hypoxia. Compounds II would be reduced by nitroreductase (NR) to the corresponding amino analogs, which would be subsequently fragmented into VNP40101M and a quinone-imine methide. NR, an enzyme isolated from E. Coli or Bacillus spp., is widely used in ADEPT (antibody-directed enzyme prodrug therapy) or GDEPT (gene-directed enzyme prodrug therapy) for cancer therapy (Anlezark, et al. WO93/08288, 1993).
FIG. 4 illustrates the use of peptidases to generate VNP40101 M intratumorally. Cleavage of compounds derived from conjugation of glutamyl residues to appropriately substituted phenols and aromatic amines by carboxypeptidases such as carboxypeptidase G2 (CPG2) and carboxypeptidase A (CPA) has been shown previously. CPG2, an enzyme isolated from Pseudomonas, is capable of removing glutamate residues from folates and methotrexate. It has been employed in the ADEPT or GDEPT system to activate prodrugs containing glutamate residues (Bagshawe, et al. WO88/07378, 1988; Springer, et al. U.S. Pat. No. 6,025,340, 2000; Springer, et al. U.S. Pat. No. 6,004,550, 1999). CPA from bovine pancreas readily cleaves drug α-peptides (derivatives in which an amino acid is linked to the α-carboxyl group of the glutamate moiety). Also, it has been employed in the ADEPT (Wolfe, et al. Bioconjug Chem. 1999, 10: 38; Huennekens, Adv Enzyme Regul. 1997, 37: 77; and Vitols, et al. Cancer Res. 1995, 55: 478). As shown in FIG. 4, Compounds III and Compounds IV would be cleaved by the corresponding CPG2 or CPA, which may either be introduced as an antibody conjugate or a transgene (Pawelek, et al. U.S. Pat. No. 6,190,657, 2001), to form VNP40101M and a quinone methide or quinone-imine methide.